B‐1 cells constitute a distinct B cell population with unique phenotypic and functional characteristics. Although the origin of B‐1 cells remains controversial, B‐1 cells in different locations are generally considered to be part of the same pool. To determine the validity of this assumption, we examined peritoneal and splenic B‐1 cells isolated by flow cytometric cell sorting from normal mice for several features. We found that splenic B‐1 cells differ from peritoneal B‐1 cells in terms of surface antigen expression, viability ex vivo, immunoglobulin secretion in vitro, stimulated cell cycle progression, and expression of Notch family, Notch‐dependent, and Notch‐associated genes. These results indicate that splenic and peritoneal B‐1 cells are not the same and thus dispute the notion that B‐1 cells are uniform, and may suggest that different subpopulations of B‐1 cells arise separately, home individually, and/or are heavily influenced by local environmental factors.